Reporter genes are used in studies of biological systems to facilitate measurement of gene activity. Generally, reporter genes are exogenous gene sequences that are introduced into cells using techniques such as transfection. Reporter genes may include an expressible region and a regulatory region. The expressible region generally encodes a readily quantifiable protein or protein activity, for example, an enzyme such as chloramphenicol acetyltransferase or beta-galactosidase, or a fluorescent protein, such as green fluorescent protein. The regulatory region generally regulates expression of the expressible region and typically includes a control element or set of control elements that mimics regulation of an endogenous gene or set of genes.
Reporter genes may be used as targets to analyze the activity of specific effector proteins. For example, cells may be engineered to express a specific receptor of interest and to include a target reporter gene that responds to the receptor. In this way, the activity of the receptor may be assessed by monitoring the presence, absence, level, and/or characteristics of the reporter gene. With this system, the activity of the receptor on the target reporter gene may be analyzed in the presence of receptor modulators to determine the ability of each modulator to function as an agonist or antagonist of receptor activity. Using this approach, natural or synthetic, activating or inhibiting ligands for the receptor may be identified in drug screens, thus providing potential candidate drugs for in vivo use.
Unfortunately, the number of known or candidate receptor proteins that has been molecularly cloned has far outstripped the ability of these methods to identify modulators of these receptor proteins by studying one receptor at a time. Thus, a multiplexed system for analyzing receptor proteins in modulator screens would greatly facilitate the identification of receptor agonists and antagonists. To determine the effects of a particular compound on multiple receptors in a single well, researchers would have to engineer a specific reporter for each receptor, introduce the engineered reporters into cells, expose the cells to the compound, and quantify the amount of each reporter. The activation of receptor #1 would result in the expression of reporter #1, the activation of receptor #2 would result in the expression of reporter #2, and so on. Therefore, the effects of a single compound on multiple receptors could be determined by quantifying the amount of each reporter.
Despite the need for multiplexed receptor analysis, current reporter genes that rely on quantification of an expressed reporter protein may have limited utility in multiplexed screens. For example, the ability to distinguish and quantify efficiently a large number of reporter proteins within a single container is impractical or impossible with current technology. For example, optical methods may be limited in their ability accurately to resolve signals produced directly or indirectly by more than two or three reporter proteins. In the case of enzyme reporter proteins, distinct enzymes may require different assay conditions, distinct substrates, and thus separate assays for each enzyme activity. Due to these inadequacies, there is a need for a reporter gene system that allows multiplexed analysis of larger sets of target genes and effector proteins.